The present invention relates to a method of diagnosing metabolic bone diseases, especially osteoporosis and arthral diseases. In addition, the present invention relates to monoclonal antibodies used in the diagnosis and kits for diagnosis using the monoclonal antibodies. The present invention is useful as a method of diagnosing metabolic bone diseases, especially osteoporosis and arthral diseases, or as assay reagents for research use thereof.
Bone metabolism depends on integrated activity of osteoblasts that form bone and osteoclasts that resorb bone. In a healthy adult, the balance of bone formation and bone resorption is kept and the bone mass is maintained constant. Metabolic bone diseases are thought to develop by losing this balance. As metabolic bone diseases, osteoporosis, hypercalcemia, Pajet""s disease, renal osteodystrophy, rheumatoid arthritis and osteoarthritis etc. are known. Osteoporosis is exemplified as a typical metabolic bone disease. Osteoporosis is thought to be a disease accompanied with decrease in bone mass and shows clinical symptoms, such as bone fracture or bone pain (lumbago and/or dorsalgia) caused by decrease in bone mass. Decrease in bone mass is induced by various causes such as aging after growing period, bone metastasis, or hyperthyroidism. As a method of diagnosing osteoporosis, bone mineral mass and/or bone density are determined by an apparatus to measure physical bone mass, such as X-ray diffraction (MD method), DPA (Dual photon absorptiometry), DEXA (Dual energy X-ray absorptiometry), CXD (Computed X-ray Densitometry) and low-frequency supersonic waves. The criterion of osteoporosis using these diagnostic methods is always rectified depending on technical revolution.
The risk of bone fracture in future might be surely predicted by decrease in bone mineral mass and/or bone density. However, decrease in bone mineral mass and/or bone density is not a sole risk factor of bone fracture and risk of bone fracture is thought to increase by phenomena accompanying with aging such as decrease in elasticity of collagen fiber, qualitative deterioration of bone structure, lowered muscular strength. At present risk factor except lowered muscular strength can not be measured non-invasively and non-invasive measurement is an important object to be solved in future. Further, decrease in bone mineral mass and/or bone density is just a result of losing the balance of bone metabolism and neither a cause of the disease or a diagnostic parameter thereof.
As supplement covering these defects of measurement of bone density, measurement of serum level and/or urinary excretion of factors regulating bone metabolism (parathyroid hormone (PTH), active form of vitamin D3 and calcitonin etc.), various kinds of factor released from bone tissue accompanying with bone remodeling (bone alkaline phosphates, acid phosphatase, pyridinoline, deoxypyridinoline, type-I procollagen peptide, osteocalcin etc.) are tried to use to diagnose the disease. These factors would reflect of bone metabolic state at the time of measurement and are expected as an early parameter of bone loss and the extent thereof. However, as for these markers of bone metabolism, there are still problems, for example, they do not express local bone metabolic change, they can be affected by diet or circadian rhythm, so that changes in the level of these above factors do not necessarily reflect specific changes in bone metabolism. From these situations, development of highly specific and precise measurement of a novel marker involved in bone metabolism is expected for establishment of methods of suitable diagnosis, prevention and treatment of various kinds of metabolic bone diseases such as osteoporosis.
The present inventors found that osteoclastgenesis inhibitory factor (OCIF) was present in a culture medium of human fetal lung fibroblasts, IMR-90 (ATCC CCL186) and succeeded in isolation thereof. In addition, the inventors also succeeded in cloning of cDNA encoding this protein and confirmed the usefulness thereof as an agent for improving bone metabolism by evaluating pharmacological effect of recombinant OCIF (rOCIF) in vitro and in vivo (WO 96/26217). Further, the present inventors confirmed that administration of rOCIF significantly improved bone density and bone strength in various kinds of animal model of metabolic bone disease and that administration of a large amount of rOCIF also significantly increased bone mass and bone volume in a normal animal without was not any side-effect in the examination of various organs other than osseous tissue, hematology and clinical biochemistry and hemolytic cell. From the results of in vivo experiment, it was found that OCIF is a highly tissue-specific cytokine having an action only on osseous tissue. In addition, the present inventors confirmed that, in an animal cell, OCIF was secreted as homodimer form of OCIF with a molecular weight of about 120 kDa and the homodimer type of OCIF was converted into monomer form of OCIF with a molecular weight of about 60 kDa by protease processing. And as it was confirmed that both types of OCIF were present in a culture medium of human cell line (Tsuda et al. :Biochem. Biophys. Res. Commun. 234, 137-142)(1997)), it is expected that both types of OCIF are present in humor of mammal including human being.
Accordingly, for elucidating whether or not OCIF can be a novel marker of bone metabolism, it is necessary to study precisely the correlation between various kinds of metabolic bone diseases and the level of each type of OCIF or total concentration of both types of OCIF in one of patients with metabolic bone diseases. Therefore, an antibody recognizing equally both types of OCIF and an antibody recognizing only homodimer are required for the above purpose. Any anti-OCIF monoclonal antibody having such features has not been obtained yet.
Considering these situations, the present inventors eagerly studied and found monoclonal antibodies with markedly high affinity (dissociation constant thereof was less than 10xe2x88x929 M) recognizing equally both of monomer- and homodimer-types of OCIF and monoclonal antibodies recognizing specifically homodimer type of OCIF. Further, the inventors constructed a highly sensitive enzyme immunoassay kit (sandwich ELISA) using these antibodies. As the results of measurement of serum concentration of OCIF in young adults, the aged, patients with osteoporosis, hyperthyroidism and various kinds of disease including cancer using the sandwich ELISA, a high inverse-correlation was found between serum concentration of OCIF and bone density. As the results of measurement of the concentration of OCIF in synovial fluid of patients with arthrosis such as rheumatoid arthritis, osteoarthritis, trauma and gouty seizure etc., OCIF concentration in synovial fluid of a patient with progressed joint destruction was found to be significantly low.
OCIF was found to be useful as a novel diagnostic marker of metabolic bone disease, because the determination of OCIF in serum and synovial fluid by the present sandwich ELISA makes it to precisely predict the dynamics of bone density and the progress of joint disruption, respectively, and thereby predict the decrease in bone mass and joint disruption at early stage of these bone decreases. Accordingly, an object of the present invention is to provide a method of diagnosing metabolic bone disease, especially osteoporosis, and joint destruction caused by rheumatism, characterized by determination of the concentration of human osteoclastgenesis inhibitory factor and monoclonal antibodies used therein and a kit for measurement of OCIF using the antibodies.
The present invention relates to a method of diagnosing metabolic bone disease by determination of the concentration of osteoclastgenesis inhibitory factor (OCIF) in sample humor.
The diagnosis of the present invention is especially useful for diagnosis of osteoporosis and arthrosis. As humor, serum or synovial fluid can be used. Diagnosis of osteoporosis can be carried out by determination of serum concentration of OCIF. And diagnosis of arthrosis can be carried out by determination of the concentration of OCIF in synovial fluid. Diagnosis by the present invention is especially useful for diagnosing osteoporosis. As a humor, serum or synovial fluid can be used.
In addition, the present invention relates to monoclonal antibodies used in the diagnosis. As monoclonal antibodies, a monoclonal antibody recognizing equally both of monomer- and dimer-types of OCIF and an antibody recognizing selectively only dimer type of OCIF can be exemplified. Further monoclonal antibodies include high affinity antibodies recognizing a different epitope and having dissociation constant of less than 2xc3x9710xe2x88x927 M with antigen.
Further, the present invention relates to a kit for OCIF determination comprising these monoclonal antibodies. The diagnostic method of the present invention can be carried out by taking humor such as blood (serum), synovial fluid from an object of diagnosis and measuring OCIF by a kit for OCIF determination using the above monoclonal antibodies.
The monoclonal antibodies can be obtained by the method described below. That is, as an antigen for immunization being necessary for preparation of anti-OCIF monoclonal antibody, human OCIF isolated from culture medium of human embryonic lung fibroblasts, IMR-90 (human embryonic lung fibroblast, ATCC CCL-186) according to a method described in WO96/26217 can be also used. Recombinant human OCIF can be also used. Recombinant human OCIF can be obtained by inserting cDNA of human OCIF into an expression vector in a conventional manner and expressing it in animal cell such as CHO cell, BHK cell and Namalwa cell etc., or insect cell followed by purification. According to the method of Tsuda et al. (Biochem. Biophys. Res. Common. 234, 137-142 (1997)), monomer- and dimer-types of OCIF can be purified by reverse-phase chromatography, respectively. Further, both types of OCIF can be respectively purified by a combination of SP-Sepharose, sulfated Cellurofine and resource S column chromatography in replace of reverse-phase chromatography. Spleen cells prepared from mammal immunized with the antigen or lymphocytes immunized in vitro can be fused with myeloma cell line to make a hybridoma. Using highly purified monomer- and homodimer-types of OCIF as antigens and culture media of the above hybridoma, the cell line can be established by screening hybridoma producing an antibody recognizing equally both types of OCIF or an antibody recognizing specifically only homodimer type of OCIF followed by cloning the hybridoma. Further, the aimed antibodies can be obtained by culturing the established and stable hybridoma.
When mammal is immunized for preparation of hybridoma, though animal species is not limited, small animal such as mice or rat is generally used. In immunization, OCIF as an antigen can be diluted in physiological saline solution to suitable concentration and the solution thereof can be administered intravenously or intraperitoneally, if necessary, Freund""s complete adjuvant can be administered therewith, generally, 3-4 times at 1-2 weeks intervals in animal. On preparation of high affinity anti-OCIF monoclonal antibody (dissociation constant thereof is less than 2xc3x9710xe2x88x927), immunization can be carried out 3 times at intervals of one week and, further, immunization with antigen together with Freund""s incomplete adjuvant can be carried out another 4 times at intervals of one week in order to obtain the aimed monoclonal antibody easily and to enhance titer of OCIF in blood as much as possible. Immunized animals described above can be anatomized 3 days after the final immunization and spleen can be dissected. Splenocytes can be used as immunized cell. As myeloma cell lines derived from mice to be hybridized with the immunized cell, p3/x63-Ag8, p3-U1, NS-1, MPC-11, SP-2/0, F0, p3x63 Ag8, 653 and S194 can be exemplified. Further, as a cell line derived from rat, R-210 can be exemplified.
To produce human antibody, human lymphocytes can be immunized in vitro and cell-fused with human myeloma cells or a human lymphocyte cell line transformed by EB virus. Fusion of immunized cells with myeloma cell line can be carried out according to a conventional method, for example, the method of Koehler and Milstein et al. (Koehler et al., Nature, 256, 495-497, 1975) but electric pulse method can be also used. Immunized lymphocytes and a myeloma cell line can be mixed at a usual rate in cell number and polyethylene glycol can be added to cell culture media used generally (not including Fetal calf serum, FCS) to carry out cell fusions and fused cells (hybridoma) can be selected by culturing in HAT selective medium containing FCS.
Hybridomas producing a monoclonal antibody recognizing equally both of monomer- and homodimer-types of OCIF and an antibody recognizing selectively homodimer type of OCIF can be selected according to a method of detecting antibody such as ELISA, plaque assay, ouchterlony method or agglutination method. ELISAs using purified monomer- and homodimer-types of OCIF can detect the object antibody very easily and precisely. It was difficult to obtain high affinity antibody (dissociation constant is less than 2xc3x9710xe2x88x927M) by usual solid phase ELISA. That is, when usual solid phase is used, culture media of hybridoma (50-100 xcexcl) is placed into 96 well immunoplates coated with antigen (Nunc) to proceed the primary reaction and, then, enzyme labeling, for example, peroxidase (POD) labeled anti-mouse IgG antibody is added to proceed the secondary reaction. Then, the solution of enzyme substrate (50-100 xcexcl) is added to each well in the immunoplates to complete enzymatic reaction and absorbance in each well is determined. Culture media of hybridoma showing high absorbance can be thought not only to produce a large amount of low affinity antibody but also to produce a high affinity antibody even if productivity of the antibody is low. It is not possible to determine which case is true.
Therefore, in the present invention, in order to recognize hybridoma producing a high affinity antibody, usual solid phase ELISA was improved as described below. That is, to each well of in 96 well immunoplates coated with an antigen, human serum or bovine serum was added, followed by the addition of a small amount of culture media of hybridoma to each well to proceed the primary reaction under the presence of about 80-90% of serum. Under such conditions, hybridomas producing antibodies with low affinity for the antigen, even if producing of the antibodies are high, can be excluded. Thus, the modified solid ELISA made it possible to selectively screen by hybridomas producing antibodies with high affinity for the antigen. Using the improved solid phase ELISA, hybridomas producing antibodies recognizing equally both of monomer- and homodimer-types of OCIF as antigens and another hybridomas producing antibodies recognizing specifically homodimer type of OCIF can be selected and stable hybridoma producing each antibody can be established by cloning 3-5 times by limited dilution method. Hybridoma established like this can be subcultured by culturing method usually used and preserved by freezing, if necessary. Hybridoma can be cultured by usual method and antibody can be recovered from the culture media. Further, antibody can be recovered from ascites derived from mammal in which hybridoma is implanted intraperitoneally. Antibody in culture media or in ascites can be purified by usual method of purifying antibody such as salting out, ion exchange or gel permeation chromatography, protein A or G affinity chromatography. Obtained antibody is an antibody recognizing equally both of monomer- and homodimer-types of OCIF and an antibody recognizing selectively homodimer type of OCIF. Each antibody can be used for measurement of the amount of OCIF (monomer type of OCIF+homodimer type of OCIF) and the amount of homodimer type of OCIF. These antibodies can be labeled by radioactive isotope or enzyme and used in radioimmunoassay (RIA) or enzymeimmunoassay (ELISA) to determine the amount of OCIF (the amount of monomer type of OCIF+homodimer type of OCIF) or the amount of homodimer type of OCIF only. Especially, the antibody of the present invention recognizing selectively homodimer type of OCIF can make it clear that there are different epitope(s) in monomer- and homodimer-types of OCIF and recognize the epitope present only in homodimer type of OCIF which is absent in monomer type of OCIF.
The amount of OCIF and the amount of homodimer type of OCIF can be determined by using an antibody obtained by the present invention recognizing equally both of monomer- and homodimer-types of OCIF as a solid phase antibody, and by using radioactive isotope or enzyme-labeled labeling antibody recognizing equally both of monomer- and homodimer-types and antibody recognizing selectively only homodimer type of OCIF with radioactive isotope or enzyme as secondary antibody, respectively.
Further, when only the amount of homodimer type of OCIF is wanted to determine, as a solid phase antibody, OI-26 antibody recognizing selectively homodimer type of OCIF as described in example 6 (table 1) and, as labeled antibody, OI-19 or OI-4 antibody recognizing equally both of monomer- and homodimer-types of OCIF can be also used. By using these assay systems, the amount of OCIF or only that of homodimer type of OCIF in body fluids such as blood, urine and synovial fluid etc. or in cell culture media can be determined.
A kit of the present invention comprises (i) any one of primary antibody and secondary antibody is OI-19 or OI-26 anti body, and (ii) the other antibody is OI-4 antibody and usual combination of reagents used in usual sandwich method. That is, an immunoassay kit comprises (1) primary antibody immobilized on insoluble carrier, (2) labeled secondary antibody, (3) solubilizer, (4) washing agent, and (5) substrate and reaction stopping reagent to determine enzymatic activity in the case of enzyme labeling. As insoluble carrier, polystyrene, polyethylene, polypropylene, polyester, polyacryronitrile, fluorinated resin, crosslinked dextran, polysaccharide, latex, latex polymer containing magnetic particles plated with metal etc., paper, glass, metal, agarose and combination of the above carries can be exemplified. As the shape of insoluble carrier, tray, sphere, fiber, stick, plate, container, cell, test tube, porous filter can be used. Further, as labeling materials used for preparation of labeled antibody, enzymes, fluorescent substances, luminescent substances and radioactive substances can be advantageously used. As enzymes, peroxidase, alkaline phosphatase, xcex2-D-galactosidase, glucose oxidase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, invertase can be used. As fluorescent substances, fluorescein isothiocyanate and phycobili-protein can be used. As luminescent substances, isolucinol and lucigenin can be used. And, as radioactive substances, I125, I131, C14, H3 can be exemplified. These above examples are merely examples and anything used in immunoassay can be used.
When a labeling material is enzyme, substrate and, if necessary, color developer can be used to determine enzymatic activity. When peroxidase is used as an enzyme, H2O2 is used as a substrate and, as color developer, 2,2xe2x80x2-azinodi[3-ethylbenzthiazoline sulfonic acid] ammonium salt(ABTS), 5-aminosalicylic acid, o-phenylenediamine, 4-aminoantipyrine, 3,3xe2x80x2,5,5xe2x80x2-tetramethylbenzidine, homocevadillinic acid, and tyramine can be used.
And when alkaline phosphatase is used as an enzyme, o-nitrophenylphosphate and 4-methylumbelliferylphosphate can be used as a substrate. When xcex2-D-galctosidase is used as an enzyme, fluoroscein-di-(xcex2-D-galctopyranoside), 4-methylumbelliferyl-xcex2-D-galctopyranoside can be used as a substrate.
As a solubilizer disclosed in (3) in the above immunoassay kit, any one used usually in immunoassay can be used, and for example, phosphate buffer solution, tris-HCl buffer solution, acetic acid buffer solution with pH of 6.0-8.0 can be respected as appropriate examples. Further, as a washing agent disclosed in (4), any one used generally in immunoassay can be used. For example, physiological saline solution, phosphate buffer solution, tris-HCl buffer solution and mixed solution thereof can be exemplified. Further, to the above washing agent, nonionic surfactant such as Triton X-100, Tween 20 or Brij 35 or ionic surfactant such as sodium dodesyl sulfate or CHAPS can be added.